Methods of manufacture of immunocompatible amniotic membrane products

Abstract

Provided herein is a placental product comprising an immunocompatible amniotic membrane. Such placental products can be cryopreserved and contain viable therapeutic cells after thawing. The placental product of the present invention is useful in treating a patient with a tissue injury (e.g. wound or burn) by applying the placental product to the injury. Similar application is useful with ligament and tendon repair and for engraftment procedures such as bone engraftment.

The invention claimed is: 
     
       1. A method of generating an immunocompatible amniotic membrane, the method comprising:
 (a) isolating an amniotic membrane from a placenta; 
 (b) refrigerating the amniotic membrane in a cryopreservation medium at 2° C. to 8° C. for at least 30 minutes to deplete one or more types of immunogenic maternal cells; and 
 (c) cryopreserving the amniotic membrane in a cryopreservation medium at a freezing temperature, thereby generating the immunocompatible amniotic membrane, wherein the immunocompatible amniotic membrane comprises at least 70% viable therapeutic cells, wherein the viable therapeutic cells are native to the amniotic membrane, wherein the viable therapeutic cells comprise two or more cell types selected from mesenchymal stem cells (MSCs), fibroblasts, and epithelial cells, and wherein the immunocompatible amniotic membrane produces a non-immunogenic response in a mixed lymphocyte reaction assay. 
 
     
     
       2. The method of  claim 1 , wherein the immunogenic maternal cells are maternal leukocytes, maternal dendritic cells, maternal decidual cells, or a combination thereof. 
     
     
       3. The method of  claim 1 , wherein the freezing temperature is between −85° C. and −75° C. 
     
     
       4. The method of  claim 1 , wherein the cryopreservation medium comprises a cell-permeating cryopreservative. 
     
     
       5. The method of  claim 4 , wherein the cell-permeating cryopreservative comprises dimethylsulfoxide (DMSO). 
     
     
       6. The method of  claim 1 , wherein refrigerating the amniotic membrane in a cryopreservation medium selectively kills or inactivates functional CD14+ macrophage cells. 
     
     
       7. The method of  claim 1 , wherein the amniotic membrane is treated with an anti-TNF-α antibody. 
     
     
       8. The method of  claim 1 , wherein the amniotic membrane is treated with IL-10. 
     
     
       9. The method of  claim 1 , wherein the immunocompatible amniotic membrane generates less than about 50 pg/ml of IL-2αR. 
     
     
       10. The method of  claim 1 , wherein the immunocompatible amniotic membrane generates less than about 20 pg/ml of IL-2αR. 
     
     
       11. The method of  claim 1 , wherein the immunocompatible amniotic membrane produces a level of TNF-α release after LPS stimulation that is less than 420 pg/ml. 
     
     
       12. The method of  claim 1 , wherein the immunocompatible amniotic membrane produces a level of TNF-α release after LPS stimulation that is less than 200 pg/ml. 
     
     
       13. The method of  claim 1 , wherein the viable therapeutic cells further comprise stromal cells present at about 2,000 to about 15,000 cells per cm 2  of the immunocompatible amniotic membrane. 
     
     
       14. The method of  claim 1 , wherein the viable therapeutic cells comprise MSCs, wherein at least 40% of the MSCs are viable after a freeze-thaw cycle.